Technical note 5: Routine proteome analysis using 50 cm μPAC™ columns

Routine proteome analysis using 50 cm μPAC™ columns

The practice of bottom-up proteomics relies to a large extent on the separation performance that can be achieved with state-of-the art nano LC-MS/MS equipment. Depending on the sample complexity or the instrument time that can be dedicated to a certain sample, different LC columns and corresponding LC-MS/MS methods are often required. When aiming for comprehensive proteome analysis with deep coverage, relatively long columns (lengths up to 75 cm) are typically operated with long and shallow solvent gradients, delivering the highest chromatographic performance. This is indeed a good strategy if very complex samples need to be analyzed and when as much information as possible needs to be retrieved from these samples. However, daily routine proteome analysis often deals with much less complex samples or demands increased sample throughput, making total analysis times above 120 min undesirable or even impossible.

As an alternative to the conventional packed bed nano LC columns, PharmaFluidics offers micromachined nano LC chip columns known as micro pillar array columns (μPAC™). The inherent high permeability and low ‘on-column’ dispersion obtained by the perfect order of the separation bed makes μPAC™ based chromatography unique in its kind. The peak dispersion originating from heterogeneous flow paths in the separation bed is eliminated (no A-term contributions) and therefore components remain much more concentrated during separation (1). The freestanding nature of the pillars also leads to much lower backpressure allowing the use of very long columns with exceptional peak capacities (2).

Complementary to its landmark 200 cm long column which is ideally suited to perform comprehensive and sometimes time consuming proteome research, PharmaFluidics now also offers a 50 cm long μPAC™ column which can be used in a more routine research setting. With an internal volume of 3 μL, this column is perfectly suited to perform high throughput analyses with shorter gradient solvent times (30, 60 and 90 minute gradients) and it can be used over a wide range of flow rates, between 100 and 2,000 nL/min.

Read the full Technical Note here